Compounds, methods and compositions useful for the treatment of bovine viral diarrhea virus (BVDV) infection and hepatitis C virus (HCV) infection

ABSTRACT

The present invention relates to novel compounds and methods that are useful in treating members of the Flaviviridae family of viruses. Compounds of the present invention will have a structure according to Formulas (I)–(VI) as recited throughout the application.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 10/044,315, filed Jan. 11, 2002, now abandoned the disclosureof which is incorporated herein by reference in its entirety, whichclaims priority to U.S. Provisional Application No. 60/261,654, filedJan. 13, 2001, the disclosure of which is incorporated herein byreference in its entirety.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under grant number K08AI01728-01 and U0I-A133383 from the National Institutes of Health. TheUnited States government may have certain rights in this invention.

FIELD OF THE INVENTION

This invention relates to the treatment of bovine viral diarrhea virus(BVDV) and hepatitis C virus (HCV) infections.

BACKGROUND OF THE INVENTION

Bovine viral diarrhea virus (BVDV) is an enveloped, single-stranded,positive sense RNA virus in the genus Pestivirus and the familyFlaviviridae. Based on the presence or absence of visible cytopathiceffect when susceptible cell monolayers are infected, two pathogenicbiotypes of BVDV, referred to as cytopathic and noncytopathic, have beenidentified. Perdrizet J A in: B. P. Smith (ed), Large Animal InternalMedicine, First Edition (Mosby Press, St Louis, 731–737 (1990)). Adifferentiation is also made between biotypes of BVDV (referred to asbiotypes I and II) based on certain viral RNA sequences in the 5′untranslated region of the genome. Pellerin C, et al., Virology 203,260–268 (1994); J. F. Ridpath et al., Virology 205, 66–74 (1994).

BVDV may cause acute infection in cattle, resulting in bovinerespiratory disease, diarrhea and severe reproductive losses. Clinicalsymptoms of acute BVDV infection may range from the almost undetectableto the severe. Infection of pregnant cows and heifers may result inbreeding problems (e.g., irregular heats), abortion, premature births orthe birth of weak or stunted calves. In some cases, temporary damage toan animal's immune system may occur even when the clinical symptoms arenot apparent. In addition to the illness caused by the virus itself,infected animals are more susceptible and are more likely to suffer fromother diseases, such as pneumonia.

In addition to causing acute disease, BVDV may also establish persistentinfections. Potgieter, Vet. Clin. North Am. Food Anim. Pract 11, 501–520(1995). Persistent BVDV infections are generally established via inutero infection of a developing fetus with a noncytopathic BVDV. Theresulting animals are born immunotolerant of the particular BVDV bywhich they are infected, and may continually shed virus throughout theirlife span. While some persistently infected animals exhibit congenitalmalformations due to BVDV infection, many animals persistently infectedwith BVDV appear clinically normal. Baker, Rev. Sci. Tech 9, 25–41(1990); Bielefeldt-Ohmann, Vet. Clin. North Am. Food Anim. Pract 11,447–476 (1995). Persistently infected animals are thought to be themajor disseminators of BVDV in the cattle population.

There are more than 140 vaccines against BVDV commercially available inthe United States. Bolin, Am J. Vet Res. 46, 2476–2470 (1995).Unfortunately, vaccination does not provide complete protection againstBVDV infection, as some vaccinated cattle still become infected with thevirus. At present, there is no known cure for BVDV infection.Accordingly, a need exists for an effective treatment for BVDVinfection.

In vitro production of embryos has become a useful therapy forincreasing reproductive performance of animals and for treatinginfertility of both animals and humans. In vitro production of bovineembryos could permit the humane, world-wide transfer of genetic materialamong cattle while limiting the transmission of many pathogens. However,in vitro-produced bovine embryos are potential vectors for transmissionof BVDV. B. Avery et al., Vet Rec 132, 660 (1993); A. Bielanski et al.,Theriogenology 46, 1467–1476 (1996); T. Tsuboi et al., Vet Microbiol 49,127–134 (1996); O. Zurovac et al., Theriogenology 41, 841–853 (1994).BVDV can be introduced into the embryo production system in associationwith gametes, serum, somatic cells, cumulus oocyte complexes (COCs), andresult in contaminated in vitro fertilized (IVF) embryos or cell lines.K. V. Brock et al., J Vet Diagn. Invest 3, 99–100 (1991); C. R. Rossi etal., Am J Vet Res 41, 1680–1681 (1980); P. J. Booth et al., J ReprodFert Abstr Ser Suppl 9, 28 (1992); M. D. Fray et al., Vet Pathol 35,253–259 (1998); R. Harasawa et al., Microbiol Immunol 39, 979–985(1995); T. Shin et al., Theriogenology 53, 243 (2000). Association ofnoncytopathic BVDV with transferred IVF embryos may cause infection ofembryo recipients, early embryonic death, abortion or birth ofpersistently infected offspring.

An analogous hazard exists in human in vitro embryo production. Viraltransmission to human embryos and embryo recipients by means ofcontaminated embryo culture media has been reported. Addition of ananti-viral agent to the culture medium surrounding in vitro-producedembryos could prevent or reduce transmission of virus to the embryo orembryo recipient. P. M. Grosheide et al., Vaccine 9, 682–687 (1991); W.G. Quint et al., J Clin Microbiol 32, 1099–1100 (1994); H. C. van Os etal., Am J Obstet Gynecol 165, 152–159 (1991). Accordingly, an antiviralagent that could be added to both animal and human in vitro embryoproduction systems may have important applications.

The organization of the portion of the BVDV genome that encodes theproteins used in viral replication is very similar to that of humanhepatitis C virus (HCV), another flavivirus. S. W. Behrens et al., JVirol 72, 2364–2372 (1998). It is believed that more than 80% of theindividuals infected with HCV will eventually develop a chronic form ofthe disease. As the disease develops, the liver of the infected subjectis progressively damaged, with the symptoms generally being commensuratewith cirrhosis and liver failure (e.g., jaundice, abdominal swelling,and finally, coma). The cycle of disease from infection to significantliver damage can take 20 years or more. Liver failure due to HCV is thepresently the leading cause of liver transplants in the United States.It is suspected that there are, at present, more than 5 million peoplein the United States that are infected with HCV, and perhaps as many as200 million around the world, making HCV infection a significant publichealth threat.

The development of a vaccine for HCV infection is uncertain, due in partto the high mutation rate of the virus. Recombinant interferon alpha-2b(INTRON A®/Schering) has proved effective in some cases of chronichepatitis C. However, it has been reported that relapse occurs in atleast half the responders after the interferon alpha-2b treatment isdiscontinued. Additionally, interferon alpha-2b may exacerbatehepatocyte injury caused by autoimmune chronic active hepatitis. J. Y.N. Lau et al., Br Med J 306, 469–470 (1993). The nucleotide analogribavirin (VIRAZOLE®/ICN Pharmaceuticals) has been shown to reduceconcentrations of hepatitis C viral RNA in an infected subject, althoughat a slower rate than interferon alpha-2b. As with BVDV infection, aneed exists for an effective treatment for HCV infection.

SUMMARY OF THE INVENTION

In view of the foregoing, one aspect of the invention relates to novelcompounds that are useful in treating members of the Flaviviridae familyof viruses, such as bovine viral diarrhea virus (BVDV) infection andhepatitis C virus (HCV) infection. Compounds of the present inventionwill have a structure according to Formulas (I)–(VI), as follows:

wherein:

X₁ and X₃ are each independently selected from the group consisting ofO, S and NR₉, wherein R₉ is H or alkyl;

X₂ and X₄ are each independently CH or N;

A is selected from the group consisting of H, alkyl, aryl,

R₁, R₂, R₃, R₄ and R₅ are each independently selected from the groupconsisting of H, alkyl, alkoxy, amidine, halide, alkylhalide, nitro andamino groups;

R₆ is H, alkyl or aryl; and

R₇ and R₈ are each independently selected from the group consisting of Hand alkyl.

Additional aspects of the invention include pharmaceutical compositionscomprising a compound having a structure according to Formulas (I)–(VI),or a pharmaceutical salt thereof (i.e., an “active compound”), in apharmaceutically acceptable carrier. Pharmaceutical compositions of thepresent invention are useful in the treatment of bovine viral diseasevirus (BVDV) infection and hepatitis C virus (HCV) infection.

Certain aspects of the invention relate to methods of treating bovineviral disease virus (BVDV) infection in a subject in need of suchtreatment. The method comprises administering to the subject a compoundaccording to Formulas (I) through (VI), or a pharmaceutically acceptablesalt thereof, in an amount effective to treat bovine viral disease virus(BVDV) infection.

Other aspects of the invention relate to methods of treating hepatitis Cvirus (HCV) infection in a subject in need of such treatment. The methodcomprises administering to the subject a compound according to Formulas(I) through (VI), or a pharmaceutically acceptable salt thereof, in anamount effective to treat hepatitis C virus (HCV) infection.

A further aspect of the present invention is the use of the activecompounds described herein for the manufacture of a medicament for thetreatment of bovine viral disease virus (BVDV) infection in a subject inneed of such treatment.

Still another aspect of the present invention is the use of the activecompounds described herein for the manufacture of a medicament for thetreatment of treating hepatitis C virus (HCV) infection in a subject inneed of such treatment.

The foregoing and other aspects of the present invention are explainedin detail in the specification set forth below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates four chemical schemes useful in the synthesis ofcompounds of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention now will be described more fully hereinafter withreference to the accompanying specification and drawings, in whichpreferred embodiments of the invention are shown. This invention may,however, be embodied in many different forms and should not be construedas limited to the embodiments set forth herein. Rather, theseembodiments are provided so that this disclosure will be thorough andcomplete, and will fully convey the scope of the invention to thoseskilled in the art.

The terminology used in the description of the invention herein is forthe purpose of describing particular embodiments only and is notintended to be limiting of the invention. As used in the description ofthe invention and the appended claims, the singular forms “a”, “an” and“the” are intended to include the plural forms as well, unless thecontext clearly indicates otherwise.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. All publications, patentapplications, patents, and other references mentioned herein areincorporated by reference in their entirety.

With respect to the compounds of the Formulas (I) through (VI), as usedherein, the term “alkyl” refers to C1–10 inclusive, linear, branched, orcyclic, saturated or unsaturated (i.e., alkenyl and alkynyl) hydrocarbonchains, including for example, methyl, ethyl, propyl, isopropyl, butyl,isobutyl, tert-butyl, pentyl, hexyl, octyl, ethenyl, propenyl, butenyl,pentenyl, hexenyl, octenyl, butadienyl, propynyl, butynyl, pentynyl,hexynyl, heptynyl, and allenyl groups. The term “alkyl” specificallyincludes cycloakyl hydrocarbon chains, which as used herein refers to C3to C6 cyclic alkyl, such as cyclopropyl, cyclobutyl, cyclopentyl, andcyclohexyl. In the present invention, preferred alkyls are the loweralkyls. The term “lower alkyl” refers to C1 to C4 linear or branchedalkyl, such as methyl, ethyl, propyl, butyl, isopropyl, sec-butyl, andtert-butyl.

The term “alkyl” also encompasses substituted alkyls, which includeaminoalkyls, hydroalkyls, oxygen-substituted alkyls (i.e., alkoxygroups), and halogen-substituted alkyls (i.e., alkyl halides,polyhaloalkyls). The term “aminoalkyl,” as used herein, refers to C1 toC4 linear or branched amino-substituted alkyl, wherein the term “amino”refers to the group NR′R″, and wherein R′ and R″ are independentlyselected from H or lower alkyl as defined above, i.e., —NH₂, —NHCH₃,—N(CH₃)2, etc. The term “hydroxyalkyl” as used herein refers to C1 to C4linear or branched hydroxy-substituted alkyl, i.e., —CH₂OH, —(CH₂)₂OH,etc. The term “alkoxy” as used herein refers to C1 to C4oxygen-substituted alkyl, i.e., —OCH₃. The term “loweralkoxy,” as usedherein, refers to C1 to C4 linear or branched alkoxy, such as methoxy,ethoxy, propyloxy, butyloxy, isopropyloxy, and t-butyloxy.

The terms “halo” and “halide” have their conventional meaning and referto fluoro, chloro, bromo, and iodo groups. Preferred halo groups includechloro groups, and preferred alkyl halides of the present inventioninclude CF₃. “Nitro” groups; as used herein, have the structure —NO₂.

The term “aryl” as used herein refers to C3 to C10 cyclic aromaticgroups such as phenyl, naphthyl, and the like, and specifically includessubstituted aryl groups including but not limited to tolyl, substitutedphenyl, and substituted naphthyl. Aryl groups may be substituted withhalo, amino, nitro, and the like. Heterocyclic aromatic rings andpolycyclic aromatic groups are also included in this definition of“aryl.” Specific examples of aryl groups encompassed by the presentinvention include but are not limited to cyclopentadienyl, phenyl,furan, thiophene, pyrrole, pyran, pyridine, imidazole, isothiazole,isoxazole, pyrazole, pyrazine, pyrimidine, and the like.

The compounds of the present invention are also useful in the form oftheir pharmaceutically acceptable salt forms. Such salts may include,but are not limited to, the gluconate, lactate, acetate, tartarate,citrate, phosphate, borate, nitrate, sulfate, hydrobromide andhydrochloric salts of the compounds. Compounds of Formulas (I)–(VI) andtheir pharmaceutically acceptable salts are referred to herein as“active compounds” or “active agents.”

The compounds represented by the Formulas (I) through (VI) may be formedby synthesis procedures that are described in the Examples below, aswell as by certain methods known in the art. Some of these known methodsare set forth below in the Examples by description or by reference (thedisclosures of which are all incorporated herein by reference in theirentirety).

Examples of compounds useful in the present invention are set forth inTable 1, below. In Table 1, the A groups are as follows:

TABLE 1 Selected Compounds Of The Present Invention Compound NameFormula A X1 X2 X3 X4 R1 R2 R3 R4 R6 R7 R8 DB 456 I A3 O C NH N NH2 H HH — H H DB 457 I A3 O C NH N NO2 H H H — H H DB 458 I A1 O C NH N NO2 HH H alkyl — — DB 459 I A1 O C NH N NH2 H H H alkyl — — DB 606 V A3 O C —— OCH3 H H H H H H DB 619 VI A1 NH C — — H H H — H — — DB 673 VI A2 O C— — H H H — H — — DB 680 VI A2 O C — — CH₃ H CH₃ — H — — DB 686 VI A2 SC — — H H H — H — — DB 687 VI A2 S N — — H H H — H — — DB 700 VI A2 O C— — H H H — H — — DB 701 VI A2 O C — — CF3 H CF3 — H — — DB 705 VI A2 OC — — H H H — H — — DB 708 VI A2 O C — — Cl H Cl — H — — DB 711 VI A2 OC — — OCH₃ H OCH₃ — H — — DB 752 VI A2 S C — — CH₃ H CH₃ — H — — DB 771II A2 O C NH N H H H — H — — DB 772 II A3 O C NH N H H H — — H H

Formulas of the compounds set forth above are as follows:

As noted above, the compounds, methods and compositions of the presentinvention are useful for treating bovine viral diarrhea virus (BVDV)infections and hepatitis C virus (HCV) infections. The term bovine viraldiarrhea virus infection means any infection (e.g., acute, latent orpersistent) caused by a virus classified as a bovine viral disease virus(BVDV). As set forth above, BVDV is an enveloped, single-stranded,positive sense RNA virus in the genus Pestivirus and the familyFlaviviridae. The term bovine viral disease virus (BVDV), as usedherein, encompasses all BVDV strains and all serotypes and variantsthereof, including live, attenuated, killed or otherwise inactivatedforms. The term BVDV specifically includes cytopathic and noncytopathicstrains, and strains of both biotype I and biotype II. The term“hepatitis C virus (HCV) infection” includes any infections caused bythe hepatitis C virus (HCV), which includes all strains, serotypes andvariants of HCV.

In one embodiment of the invention, a subject is administered atherapeutically-effective amount of the compound of formulas (I) through(VI), or a pharmaceutically acceptable salt thereof. A“therapeutically-effective” amount as used herein is an amount of acompound of formulas (I) through (VI) that is sufficient to alleviate(e.g., mitigate, decrease, reduce) at least one of the symptomsassociated with BVDV or HCV infection. It is not necessary that theadministration of the compound eliminate the symptoms of BVDV or HCV, aslong as the benefits of administration of compound outweigh thedetriments. Likewise, the terms “treat” and “treating” in reference toBVDV or HCV, as used herein, are not intended to mean that the aviansubject is necessarily cured of BVDV or HCV, or that all clinical signsthereof are eliminated, only that some alleviation or improvement in thecondition of the subject is effected by administration of the compoundof Formulas (I) through (VI).

Suitable subjects of the present invention include humans and animals.When the subject is an animal, mammals are preferred, with livestock(e.g., cattle, pigs, sheep, horses) and primates (e.g., monkeys, apes)being particularly preferred. In embodiments of the present inventionwhere BVD are treated, bovine subjects (e.g., cows, bulls, calves) arepreferred. In embodiments of the present invention where HCV infectionsare treated, humans are the preferred subjects. Subjects may be adult,adolescent, juvenile, infant, or neonatal. In one embodiment of theinvention, the subject is a live embryo, and may be in utero or in vitro(in the case of an embryo being maintained for in vitro fertilization).

Subjects may be administered the compounds and compositions of thepresent invention by any suitable means. Exemplary means are oraladministration (e.g., in the form of a liquid or solid), intramuscularinjection, subcutaneous injection, and intravenous injection.Pharmaceutical formulations of the present invention comprise activecompounds of the invention in a pharmaceutically acceptable carrier.Suitable pharmaceutical formulations include those suitable forinhalation, oral, rectal, topical, (including buccal, sublingual,dermal, vaginal and intraocular), parenteral (including subcutaneous,intradermal, intramuscular, intravenous and intraarticular) andtransdermal administration. The most suitable route of administration inany given case may depend upon the anatomic location of the conditionbeing treated in the subject, the nature and severity of the conditionbeing treated, and the particular active compound which is being used.The formulations may conveniently be presented in unit dosage form andmay be prepared by any of the methods well known in the art.

In methods of the present invention where treatment is carried outduring an in vitro fertilization (IVF) procedure, the compounds may beadministered to the embryo by adding the active compound, in a suitableconcentration, to the medium in which the embryo is being obtained.

In the manufacture of a medicament according to the invention (the“formulation”), active compounds or the pharmaceutically acceptablesalts thereof (the “active compounds”) are typically admixed with, interalia, an acceptable carrier. The carrier must, of course, be acceptablein the sense of being compatible with any other ingredients in theformulation and must not be deleterious to the patient. The carrier maybe a solid or a liquid, or both, and is preferably formulated with thecompound as a unit-dose formulation, for example, a tablet, which maycontain from 0.5% to 99% by weight of the active compound. One or moreactive compounds may be incorporated in the formulations of theinvention, which formulations may be prepared by any of the well knowntechniques of pharmacy consisting essentially of admixing thecomponents, optionally including one or more accessory therapeuticingredients.

Formulations suitable for oral administration may be presented indiscrete units, such as capsules, cachets, lozenges, or tablets, eachcontaining a predetermined amount of the active compound; as a powder orgranules; as a solution or a suspension in an aqueous or non-aqueousliquid; or as an oil-in-water or water-in-oil emulsion. Suchformulations may be prepared by any suitable method of pharmacy whichincludes the step of bringing into association the active compound and asuitable carrier (which may contain one or more accessory ingredients asnoted above). In general, the formulations of the invention are preparedby uniformly and intimately admixing the active compound with a liquidor finely divided solid carrier, or both, and then, if necessary,shaping the resulting mixture. For example, a tablet may be prepared bycompressing or molding a powder or granules containing the activecompound, optionally with one or more accessory ingredients. Compressedtablets may be prepared by compressing, in a suitable machine, thecompound in a free-flowing form, such as a powder or granules optionallymixed with a binder, lubricant, inert diluent, and/or surfaceactive/dispersing agent(s). Molded tablets may be made by molding, in asuitable machine, the powdered compound moistened with an inert liquidbinder. Formulations for oral administration may optionally includeenteric coatings known in the art to prevent degradation of theformulation in the stomach and provide release of the drug in the smallintestine.

Formulations of the present invention suitable for parenteraladministration comprise sterile aqueous and non-aqueous injectionsolutions of the active compound, which preparations are preferablyisotonic with the blood of the intended recipient. These preparationsmay contain anti-oxidants, buffers, bacteriostats and solutes whichrender the formulation isotonic with the blood of the intendedrecipient. Aqueous and non-aqueous sterile suspensions may includesuspending agents and thickening agents. The formulations may bepresented in unit\dose or multi-dose containers, for example sealedampoules and vials, and may be stored in a freeze-dried (lyophilized)condition requiring only the addition of the sterile liquid carrier, forexample, saline or water-for-injection immediately prior to use.Extemporaneous injection solutions and suspensions may be prepared fromsterile powders, granules and tablets of the kind previously described.For example, in one aspect of the present invention, there is providedan injectable, stable, sterile composition comprising a compound ofFormula (I)–Formula (VI), or a salt thereof, in a unit dosage form in asealed container. The compound or salt is provided in the form of alyophilizate which is capable of being reconstituted with a suitablepharmaceutically acceptable carrier to form a liquid compositionsuitable for injection thereof into a subject. The unit dosage formtypically comprises from about 10 mg to about 10 grams of the compoundor salt. When the compound or salt is substantially water-insoluble, asufficient amount of emulsifying agent which is physiologicallyacceptable may be employed in sufficient quantity to emulsify thecompound or salt in an aqueous carrier.

Further, the present invention provides liposomal formulations of thecompounds disclosed herein and salts thereof. The technology for formingliposomal suspensions is well known in the art. When the compound orsalt thereof is an aqueous soluble salt, using conventional liposometechnology, the same may be incorporated into lipid vesicles. In such aninstance, due to the water solubility of the compound or salt, thecompound or salt will be substantially entrained within the hydrophiliccenter or core of the liposomes. The lipid layer employed may be of anyconventional composition and may either contain cholesterol or may becholesterol-free. When the compound or salt of interest iswater-insoluble, again employing conventional liposome formationtechnology, the salt may be substantially entrained within thehydrophobic lipid bilayer which forms the structure of the liposome. Ineither instance, the liposomes which are produced may be reduced insize, as through the use of standard sonication and homogenizationtechniques.

Of course, the liposomal formulations containing the pharmaceuticallyactive compounds identified with the methods described herein may belyophilized to produce a lyophilizate which may be reconstituted with apharmaceutically acceptable carrier, such as water, to regenerate aliposomal suspension.

In addition to the active compounds, the pharmaceutical formulations maycontain other additives, such as pH-adjusting additives. In particular,useful pH-adjusting agents include acids, such as hydrochloric acid,bases or buffers, such as sodium lactate, sodium acetate, sodiumphosphate, sodium citrate, sodium borate, or sodium gluconate. Further,the compositions may contain microbial preservatives. Useful microbialpreservatives include methylparaben, propylparaben, and benzyl alcohol.The microbial preservative is typically employed when the formulation isplaced in a vial designed for multidose use. Of course, as indicated,the pharmaceutical formulations of the present invention may belyophilized using techniques well known in the art.

Pharmaceutical formulations of the present invention may comprisecompounds of the present invention in lyophilized form. Alternatively,pharmaceutical formulations of the present invention may comprisecompounds of the present invention in a pharmaceutically acceptablecarrier. Such pharmaceutical formulations are generally made by admixingthe compounds described herein with a pharmaceutically acceptablecarrier. Pharmaceutically acceptable carriers are preferably liquid,particularly aqueous, carriers, the selection of which are known in theart. For the purpose of preparing such formulations, the compound may bemixed in a buffered saline (e.g., pH 6 to 8) or conventional culturemedia. The formulation may be stored in a sterile glass container sealedwith a rubber stopper through which liquids may be injected andformulation withdrawn by syringe.

With respect to all the methods described herein, a therapeuticallyeffective dosage of any specific compound, the use of which is in thescope of present invention, may vary somewhat from compound to compoundand subject to subject, and will depend upon the condition of thesubject and the route of delivery. A dosage from about 1 mg/kg to about15 mg/kg of subject body weight, or about 20 mg/kg of subject bodyweight, or even about 25 mg/kg of subject body weight may be employedfor intravenous injection or oral administration.

The concentration of the compound of the present invention or apharmaceutically acceptable salt thereof in a formulation of the presentinvention may be determined by the skilled artisan and will varyaccording to certain conditions, including the characteristics ofsubject being treated (e.g., species, age, weight), the severity andtype of the infecting virus or the strain that the subject is beingvaccinated against, the dosage form being used, and the like.

The compounds of the present invention may be administered inconjunction with other antiviral compounds, as may be determined by theskilled artisan.

The present invention is explained in greater detail in the Exampleswhich follow. These examples are intended as illustrative of theinvention, and are not to be taken as limiting thereof.

EXAMPLES 1–12 Synthesis of Inventive Compounds

In the following Examples, compound numbers (compounds 2, 5, 5a, etc.)refer to compounds with structures that are set forth in FIG. 1.

Example 1

General Methodology: Chemical Synthesis and Analysis

Melting points were determined with a MEL-TEMP® 3.0 capillary meltingpoint apparatus and are uncorrected. ¹H nuclear magnetic resonancespectra were recorded on a Varian Unity+300 or a Varian VRX 400instrument, with peak assignments relative to residual DMSO (2.49 ppm)or CHCl₃ (7.24 ppm). Mass spectra were recorded on a VG Instruments70-SE spectrometer at the Georgia Institute of Technology, Atlanta, Ga.Elemental analyses were performed by Atlantic Microlab, Norcross, Ga.All final compounds were dried in vacuo (oil pump) at 50–60° C. for atleast 36 hours before elemental analysis. Unless otherwise stated, allreagent chemicals and solvents (including anhydrous solvents) werepurchased from Aldrich Chemical Co., Fisher Scientific, or LancasterSynthesis and used as received. Acetonitrile (CaH₂), triethylamine(CaH₂), and ethanol (Mg/I₂) were distilled from the indicated dryingagent. 2,6-Dimethyl-4-nitrobromobenzene and S-(2-Naphthylmethyl)thioacetimidate were prepared according to the literature. See B. M.Wepster, Rec. Trav. Chim. 73, 809–818 (1954); D. N. Kravtsov, J.Organometal. Chem. 36, 227–237 (1972); B. G. Shearer et al., TetrahedronLett. 38, 179–182 (1997).

Example 2 Preparation of 2,5-bis(4-nitrophenyl)furans

The following representative procedures are variations of a generalprocedure previously described in A. Kumar et al., Heterocyclic Comm. 5,301–304 (1999).

2,5-Bis(2-methyl-4-nitrophenyl)furan (Compound 2b). To a solution of2-bromo-5-nitrotoluene (4.32 g, 20 mmol) and tetrakis(triphenylphospine) palladium (0) (0.40 g) in anhydrous 1,4-dioxane (50ml) was added 2,5-bis (tri-n-butylstannyl) furan (6.46 g, 10 mmol) andthe mixture was heated overnight under nitrogen at 95–100° C. Theresulting orange suspension was diluted with hexanes (15 ml), cooled toroom-temperature, and filtered to give, after rinsing with hexanes, anorange solid (3.10 g), mp 241–243° C. The product was recrystallizedfrom DMF (100 ml) to give a bright orange fluffy solid (2.87 g, 85%), mp242–243° C. ¹H NMR (DMSO-d₆): 2.69 (s, 6H), 7.31 (s, 2H), 8.12 (m, 4H),8.23 (s, 2H). Anal. Calcd. for C₁₈H₁₄N₂O₅ (338.31): C, H, N.

2,5-Bis(4-nitrophenyl)furan (Compound 2a). Yield: 88%; orange fluffysolid; mp 269–270° C. (not recrystallized), lit. mp 270–272° C., Ling,C. et al., J. Am. Chem. Soc. 1994, 116, 8784–8792.

2,5-Bis(2-methoxy-4-nitrophenyl)furan (Compound 2c). Yield: 77%; brightorange granular solid; mp 308–310° C. (DMF). ¹H NMR (DMSO-d₆): 4.10 (s,6H), 7.37 (s, 2H), 7.90 (s, 2H), 7.94 (d, 2H), 8.22 (d, 2H). Anal.Calcd. for C₁₈H₁₄N₂O₇.0.1H₂O (372.11): C, H, N.

2,5-Bis(2-chloro-4-nitrophenyl)furan (Compound 2d). Yield: 71%; fluffyorange solid; mp 247–247.5° C. (DMF/MoeOH). ¹H NMR (DMSO-d₆): 7.70 (s,2H), 8.29 (dd, J=8.8, 2.2 Hz, 2H), 8.36 (d, J=8.8 Hz, 2H) 8.43 (d, J=2.2Hz, 2H). Anal. Calcd. for C₁₆H₈Cl₂N₂O₅ (379.15): C, H, N.

2,5-Bis(4-nitro-2-trifluoromethylphenyl)furan (Compound 2e). Yield: 74%;fluffy golden needles; mp 158.5–159° C. (EtOH). ¹H NMR (DMSO-d₆): 7.38(s, 2H), 8.24 (d, J=8.7 Hz, 2H), 8.57 (d, J=2.4 Hz, 2H), 8.62 (dd,J=8.6, 2.4 Hz, 2H). Anal. Calcd. for C₁₈H₈F₆N₂O₅ (446.26): C, H, N.

2,5-Bis(2,6-dimethyl-4-nitrophenyl)furan (Compound 2f). Yield: 65%;yellow needles; mp 156.5–157.5° C. (DMF/EtOH/H₂O). ¹H NMR (DMSO-d₆):2.34 (s, 12H), 6.85 (s, 2H), 8.04 (s, 4H). Anal. Calcd. for C₂₀H₁₈N₂O₅(366.36): C, H, N.

Example 3 Preparation of 2,5-bis(4-aminophenyl)furans

The following procedures are representative.

2,5-Bis(4-amino-2-methylphenyl)furan (Compound 3b). To a suspension ofthe bis-nitro derivative 2b (2.87 g) in EtOAc (90 ml) and dry EtOH (10ml) was added Pd/C (10%) (0.40 g) and the mixture was hydrogenated on aParr apparatus at an initial pressure of ˜50 psi. After the uptake ofhydrogen subsided (generally 3–6 hours), the resulting solution wasfiltered over Celite and the pale yellow to colorless filtrate wasconcentrated in vacuo to near dryness to give, after dilution withhexanes, the pure diamine as a pale yellow/green solid (2.17 g, 91%), mp174–176° C., which required no purification. ¹H NMR (DMSO-d₆): 2.33 (s,6H), 5.15 (br s, 4H), 6.42 (s, 2H), 6.46 (m, 4H), 7.35 (d, 2H). MS (EI):m/z 278 (M⁺).

2,5-Bis(4-aminophenyl)furan (Compound 3a). Yield: 94%; pale green/tansolid; mp 218–221° C., lit⁴⁶ mp 213–216° C. MS (EI): m/z 250 (M⁺).

2,5-Bis(4-amino-2-methoxyphenyl)furan (Compound 3c). The original oilwas reconcentrated with benzene to give a yellow/tan solid which wastriturated with ether.

Yield: 79%; mp 201–202.5° C. ¹H NMR (DMSO-d₆): 3.80 (s, 6H), 5.25 (br s,4H), 6.24 (dd, J=8.3, 2.0 Hz, 2H), 6.30 (d, J=1.9 Hz 2H), 6.56 (s, 2H),7.48 (d, J=8.4 Hz, 2H). MS (EI): m/z 310 (M⁺).

2,5-Bis(4-amino-2-trifluoromethylphenyl)furan (Compound 3e). Originalred oil crystallized from EtOAc/hexanes in two crops as a red/orangesolid. Combined yield: 81%; mp (first/major crop) 89.5–91° C.; mp(second crop) 91.5–92° C. ¹H NMR (DMSO-d₆): 5.79 (br s, 4H), 6.52(s,2H), 6.82 (dd, J=8.4, 2.4 Hz, 2H), 6.98(d, J=2.2 Hz, 2H), 7.43 (d, J=8.4Hz, 2H). MS (EI): m/z 386 (M⁺).

2,5-Bis(4-amino-2,6-dimethylphenyl)furan (Compound 3f). Yield: 99%;white fluffy solid; mp 144.5–146° C. ¹H NMR (DMSO-d₆): 2.01 (s, 6H),5.06 (br s, 4H), 6.24 (s, 2H), 6.29 (s, 4H). MS (EI): m/z 306 (M⁺).

2,5-Bis(4-amino-2-chlorophenyl)furan (Compound 3d). To a suspension ofthe corresponding bis-nitro derivative 2d (1.22 g, 3.2 mmol) in dry EtOH(100 ml) and DMSO (20 ml) was added SnCl₂.2H₂O (5.80 g, 25.7 mmol) andthe mixture was heated under nitrogen at 80° C. After 4–5 hours, TLCshowed that starting material had been consumed, and thus the mixturewas cooled, neutralized with NaOH (aq), and extracted with EtOAc. Theextract was washed with water, brine, then dried (Na₂SO₄) andconcentrated. The resulting oil was crystallized from benzene/hexanewith partial concentration to give a light brown solid (0.74 g, 71%), mp191.5–193° C. Catalytic hydrogenation was not explored. ¹H NMR(DMSO-d₆): 5.60 (br s, 4H), 6.61 (dd, J=8.6, 2.2 Hz, 2H), 6.68 (d, J 2.2Hz 2H), 6.82 (s, 2H), 7.56 (d, J=8.6 Hz, 2H). MS (EI): m/z 318 (M⁺).

Example 4 Preparation of 2,5-bis(4-N,N′-di-BOC-guanidinophenyl)furanderivatives

The following procedures are representative.

2,5-Bis(4-N,N′-di-BOC guanidinophenyl)furan (Compound 4a). To aroom-temperature solution of 2,5-bis(4-aminophenl)furan (0.626 g, 2.5mmol) and 1,3-bis(tert-butoxycarbonyl)-2-methyl-2-thiopseudourea (1.56g, 5.3 mmol) in anhydrous DMF was added triethylamine (1.59 g, 15.7mmol) followed by mercury (II) chloride (1.57 g, 5.8 mmol) and theresulting suspension was stirred at room-temperature for 22 hours. Afterdiluting with CH₂Cl₂ and sodium carbonate solution, the suspension wasfiltered over Celite and the filtrate was washed well with water (3×)and finally with brine. After drying (Na₂SO₄), the solvent was removedin vacuo and the residue was diluted with MeOH to give the BOC-protectedbis-guanidine as a pale yellow solid. The collected product was purifiedby reprecipitation from CH₂Cl₂/MeOH to give a fluffy yellow solid (1.25g, 68%), mp>400° C. dec. ¹H NMR (CDCl₃): 1.50 and 1.53 (2s, 36H), 6.65(s, 2H), 7.66 (s, 8H); 10.38 (br s, 2H), 11.61 (br s, 2H).

2,5-Bis(2-methyl-4-N,N′-di-BOCguanidinophenyl)furan (Compound 4b).Yellow solid, mp>250° C. dec. Yield: 62%. ¹H NMR (CDCl₃): 1.51 and 1.52(2 s, 36H), 2.53 (s, 6H), 6.60 (s, 2H), 7.40 (s, 2H), 7.62 (d, 2H), 7.74(d, 2H), 10.34 (s, 2H), 11.62 (br s, 2H).

2,5-Bis(2-methoxy-4-N,N′-di-BOCguanidinophenyl)furan (Compound 4c).Yellow solid, mp>300° C. dec. Yield: 79%. ¹H NMR (CDCl₃): 1.50 and 1.53(2 s, 36H), 3.95 (s, 6H), 6.95 (s, 2H), 7.13 (d, 2H), 7.59 (s, 2H), 7.86(d, 2H), 10.36 (s, 2H), 11.55 (br s, 2H).

2,5-Bis(2-chloro-4-N,N′-di-BOCguanidinophenyl)furan (Compound 4d). Paleyellow/tan solid, mp>400° C. dec. Yield: 63%. ¹H NMR (CDCl₃): 1.52 (s,36H), 7.17 (s, 2H), 7.63 (dd, 2H), 7.79 (d, 2H), 7.38 (dd, 2H), 10.43(s, 2H), 11.59 (br s, 2H).

2,5-Bis(2-trifluoromethyl-4-N,N′-di-BOC guanidinophenyl)furan (Compound4e). Bright orange solid. Yield: 88%. ¹H NMR (CDCl₃): 1.51 and 1.53 (2s, 36H), 6.77 (s, 2H), 7.82 (d, 2H), 7.94 (s, 2H), 8.00 (d, 2H), 10.52(s, 2H), 11.59 (br s, 2H).

2,5-Bis(2,6-dimethyl-4-N,N′-di-BOC guanidinophenyl)furan (Compound 4f).Pale yellow/off-white solid, mp>300° C. dec. Yield: 89%. ¹H NMR (CDCl₃):1.51 and 1.53 (2 s, 36H), 2.23 (s, 12H), 6.31 (s, 2H), 7.33 (s, 4H),10.27 (s, 2H), 11.63 (br s, 2H).

Example 5

Deprotection: of N,N′-di-BOC guanidines

The following procedures are representative, and are further illustratedin FIG. 1.

2,5-Bis(4-guanidinophenyl)furan dihydrochloride (Compound 5a). Asolution of the corresponding N,N′-di-BOCguanidine (1.19 g, 1.62 mmol)in CH₂Cl₂ (15 ml) was diluted with dry EtOH (10 ml) and saturated atice-water bath temperature with anhydrous HCl. The solution was thenstirred at room-temperature for 2–3 days (drying tube), with the productslowly precipitating (shorter reaction times generally gave incompletedeprotection). The resulting suspension was concentrated to neardryness, with the solid then taken up in hot EtOH. After filtering toclarify, the solution was concentrated to near dryness to give asuspension, which was diluted with ether and collected to yield, afterdrying in vacuo at 50–60° C. for 2 days, the bis-guanidinedihydrochloride as an off-white/tan solid (0.66 g, quantitative),mp>300° C. dec. ¹H NMR (DMSO-d₆): 7.12 (s, 2H), 7.31 (d, 4H), 7.58 (brs, 8H), 7.86 (d, 4H), 10.09 (br S, 2H). MS (FAB, thioglycerol): m/z335.3 (MH⁺, 100). Anal. Calcd. for C₁₈H₁₈N₆O.2HCl.0.25EtOH (407.30): C,H, N.

2,5-Bis(4-guanidino-2-methylphenyl)furan dihydrochloride (Compound 5b).Tan solid, mp 265–271° C. dec. ¹H NMR (DMSO-d₆): 2.53 (s, 6H), 6.93 (s,2H), 7.17 (m, 4H), 7.56 (br S, 8H), 7.82 (d, 2H), 10.06 (br S, 2H). MS(FAB, thioglycerol): m/z 363.3 M, 100). Anal. Calcd. forC₂₀H₂₂N₆O.2HCl.1.5H₂O.0.66EtOH (496.93): C, H, N.

2,5-Bis(4-guanidino-2-methoxyphenyl)furan dihydrochloride (Compound 5c).Light brown solid. ¹H NMR (DMSO-d₆): 3.95 (s, 6H), 6.92 (dd, 2H), 6.99(d, 2H), 7.02 (s, 2H), 7.58 (br S, 8H), 7.95 (d, 2H), 10.08 (br S, 2H).MS (EI): m/z 352 (M⁺-NH₂CN, 38.0), 310 (100), 267 (38.9), 251 (8.8), 155(18.7). Anal. Calcd. for C₂₀H₂₂N₆O₃.2HCl.1.0H₂O.0.33EtOH (500.57): C, H,N.

2,5-Bis(2-chloro-4-guanidinophenyl)furan dihydrochloride (Compound 5d).Tan solid, mp 300–304° C. dec. ¹H NMR (DMSO-d₆): 7.31 (s, 2H), 7.33 (d,2H), 7.47 (s, 2H), 7.72 (br S, 8H), 8.04 (d, 2H). MS (DCI, ammonia): m/z365, 363, 361 (MH⁺-NH₂CN, 8, 52, 78), 323, 321, 319 (11, 66, 100). Anal.Calcd. for C₁₈H₁₆Cl₂N₆O.2HCl.0.5H₂O (485.21): C, H, N, Cl.

2,5-Bis (4-guanidine-2-trifluoromethylphenyl) furan dihydrochloride(Compound 5e). Orange/red solid. ¹H NMR (DMSO-d₆): 6.99 (s, 2H), 7.63(d, 2H), 7.69 (s, 2H), 7.79 (br S, 8H), 7.91 (d, 2H), 10.37 (br S, 2H).MS (CI, isobutane): m/z 471 (MH⁺, 14), 429 (100), 387 (19). Anal. Calcd.for C₂₀H₁₆F₆N₆O.2HCl.0.67H₂O.0.67EtOH (586.24): C, H, N.

2,5-Bis(4-guanidino-2,6-dimethylphenyl)furan dihydrochloride (Compound5f). Off-white solid. ¹H NMR (DMSO-d₆): 2.20 (s, 12H), 6.56 (s, 2H),7.01 (s, 4H), 7.57 (br s, 8H), 10.09 (br S, 2H). MS (FAB, thioglycerol):m/z 391.2 (MH⁺, 100). Anal. Calcd. for C₂₂H₂₆N₆O.2HCl.0.5H₂O (472.41):C, H, N.

Example 6 Preparation of2-[5(6)-Amidino-2-benzimidazoyl]-5-(4-nitrophenyl)furan

A mixture of 5-(4-nitrophenyl)furfural (0.651 g, 0.003 mol),4-amidino-1,2-phenylenediamine (0.614 g, 0.003 mol) and 1,4-benzoquinone(0.324 g, 0.003 mol) in 40 ml of ethanol (under nitrogen) was heated atreflux for 8 h. The volume of the reaction mixture was reduced to 20 mlunder reduced pressure, cooled and the resultant solid was collected byfiltration. The solid was washed with cold ethanol and ether. Theproduct was dried to yield the mono hydrochloride salt 0.8 g (70%). Themono salt (0.65 g) was dissolved in 120 ml of ethanol and acidified withHCl-saturated ethanol and after standing overnight in a refrigerator theresultant solid was filtered, washed with ether and dried for 24 h in avacuum oven at 70° C. to yield 0.6 g (85%) mp 300° C. ¹H NMR (DMSO-d₆):9.3 (br s, 2H), 9.09 (br s, 2H), 8.33 (d, J=7.6 Hz, 2H), 8.20 (d, J=7.6Hz 2H), 8.19 (s, 1H), 7.79 (d, J=8.4 Hz, 1H), 7.73 (d, J=8.4 Hz, 1H),7.56 (d, J=3.6 Hz, 1H), 7.51 (d, J=3.6 Hz 1H). ¹³C NMR (DMSO-d₆): 165.9,152.6, 146.4, 145.4, 145.3, 141.6, 138.7, 134.7, 124.6, 124.0, 122.1,121.5, 116.0, 114.6,114.0, 111.9. FABMS m/e 348(M⁺+1). Anal. Calcd forC₁₈H₁₃N₅O₃.2HCl.: C, 51.44; H, 3.59; N, 16.66. Found: C, 51.24; H, 4.03;N, 16.92.

Example 7 Preparation of2-[5(6)-amidino-2-benzimidazoyl]-5-(4-aminophenyl)furan

The above nitro analog (0.5 g, 0.0013 mol) and 0.3 g of 10% Pd/C in 130ml of methanol was subjected to hydrogenation at 50 psi for 4 h. Thecatalyst was removed by filtration over filteraid and the solvent wasremoved under reduced pressure. The solid was taken up in methanolicHCl, warmed on a water bath for 0.5 h and the solvent was removed underreduced pressure. The residue was treated with ether and the solid wascollected by filtration and dried under vacuum at 75° C. for 12 h toyield 0.44 g (73%) mp>360° C. ¹H NMR (DMSO-d₆/D₂O): 8.07 (d, J=1.6 Hz,1H), 7.74(d, J=8.4 Hz, 2H), 7.66 (dd, J=1.6 and 8.4 Hz 2H), 7.39 (d,J=3.6 Hz, 1H), 6.91 (d, J=3.6 Hz, 1H), 6.89 (d, J=8.4 Hz, 2H). ¹³C NMR(DMSO-d₆/D₂O): 166.2, 156.7, 145.8, 142.0, 141.0, 138.1, 126.2, 123.2,122.3, 119.2, 116.6, 116.0, 115.1, 107.5. FABMS m/e 318(M⁺+1). Anal.Calcd. for C₁₈H₁₅N₅O₃HCl.2H₂O: C, 43.59; H, 6.09; N, 16.92. Found: C,43.71, H, 6.01, N, 16.81.

Example 8 Preparation of 2-[5(6)-{2-imidazolinyl}-2-benzimidazoyl]-5-(4-nitrophenyl) furan

A mixture of 5-(4-nitrophenyl)furfural (0.434 g, 0.002 mol),4-(2-imidazolinyl)-1,2-phenylenediamine hydrochloride hydrate (0.461 g,0.002 mol) and 1,4-benzoquinone (0.216 g, 0.002 mol) in 40 ml of ethanol(under nitrogen) was heated at reflux for 8 h. The volume of thereaction mixture was reduced to 20 ml under reduced pressure, cooled andthe resultant solid was collected by filtration. The solid was washedwith cold ethanol and ether. The product was dried to yield 0.52 g(63%). The compound was dissolved in 200 ml of ethanol and acidifiedwith HCl-saturated ethanol and was stirred at room temperature for 3 h.The mixture was cooled on ice and the solid was filtered, washed withether and dried for 24 h in a vacuum oven at 75° C. to yield 0.51 g(90%) mp>300° C. ¹H NMR (DMSO-d₆/D₂O): 8.31 (d, J=. 8.4 Hz, 2H), 8.30(s, 1H), 8.15 (d, J=8.4 Hz, 2H), 7.8:1 (s, 2H), 7.52 (d, J=4.0 Hz, 1H),7.46 (d, J=4.0 Hz, 1H), 4.03 (s, 4H). ¹³C NMR (DMSO-d₆/D₂O): 165.6,153.1, 146.8, 145.7, 145.2, 134.7, 124.9, 124.2, 122.8, 116.9, 115.8,115.1, 115.0, 112.1, 105.6, 104.7, 44.2. FABMS m/e 374 (M⁺+1). Anal.Calcd for C₂₀H₁₅N₅O₃.2HCl: C, 53.82; H, 3.88; N, 15.69. Found: C, 53.94;H, 3.93; N, 15.84.

Example 9 Preparation of 2-[5 (6)-{2-imidazolinyl}-2-benzimidazoyl]-5-(4-aminophenyl)furan

The mono hydrochloride salt of the above nitro analog (0.5 g, 0.0013mol) and 0.2 g of 10% Pd/C in 130 ml of methanol was subjected tohydrogenation at 50 psi for 4 h. The catalyst was removed by filtrationover filteraid, washed with warm methanol. The solvent volume wasreduced to approximately half under reduced pressure. The flaskcontaining the solution was placed in an ice bath and saturated with HClgas. The mixture was stirred at room temperature for 4 h and treatedwith dry ether and the solid was collected by filtration. The solid wasdried under vacuum at 75° C. for 24 h to yield 0.55 g (86%) mp>300° C.¹H NMR (DMSO-d₆/D₂O): 8.24 (d, J=1.2 Hz 1H), 7.88 (d, J=8.0 Hz, 2H),7.80 (s, 2H), 7.51(d, J=3.6 Hz, 1H), 7.21 (d, J=8.4 Hz, 2H), 7.10 (dd,J=1.2,3.6 Hz, 1H), 4.0 (s, 4H). ¹³C NMR (DMSO-d₆/D₂O): 165.8, 156.4,145.8, 142.0, 140.9, 137.9, 126.2, 123.7, 121.0, 117.0, 116.8, 115.3,108.5, 44.6. FABMS m/e 344(M⁺+1). Anal. Calcd for C₂₀H₁₇N₅O.3HCl₂.1H₂O:C, 48.96; H, 4.97; N, 14.27. Found: C, 48.58; H, 4.32; N, 14.27.

Example 10 Preparation of 2-[5(6)-{N-isopropylamidino}-2-benzimidazoyl]-5-(4-nitrophenyl)furan

A mixture of 5-(4-nitrophenyl)furfural (0.434 g, 0.002 mol),4-N-isopropylamidino-1,2-phenylenediamine hydrochloride hydrate (0.493g, 0.002 mol) and 1,4-benzoquinone (0.216 g, 0.002 mol) in 40 ml ofethanol (under nitrogen) was heated at reflux for 6 h. The volume of thereaction mixture was reduced to about 15 ml under reduced pressure, themixture was cooled and the resultant solid was collected by filtrationto yield the mono hydrochloride salt 0.66 g (80%). The mono salt wasdissolved in 100 ml of ethanol and acidified with HCl-saturated ethanoland after cooling in an ice bath the resultant solid was filtered,washed with ether and dried for 24 h in a vacuum oven at 75° C. to yield0.7 g (91%) mp>300° C. ¹H NMR (DMSO-d₆/D₂O): 8.26 (d, J=8.8 Hz, 2H),8.11 (d, J=8.8 Hz 2H), 8.01 (d, J=1.2 Hz, 1H), 7.77 (d, J=8.8 Hz, 1H),7.59 (dd, J=1.2, 8.8 Hz, 1H), 7.50(d, J=7.6 Hz, 1H), 7.42 (d, J=7.6 Hz1H), 4.04 (septet, J=6.8 Hz, 1H), −1.3(d, J=6.8 Hz, 6H). ¹³C NMR(DMSO-d₆): 162.7, 153.8, 147.2, 145.2, 144.8, 140.7, 138.2, 135.2,125.4, 124.7, 124.0, 123.5, 116.3, 115.9, 115.3, 112.6, 45.6, 21.4.FABMS m/e 376(M⁺+1). Anal. Calcd for C₂₁H₁₉N₅O₃.2HCl.2.0H₂O: C, 49.71;H, 5.16; N, 13.80. Found: C, 49.65; H, 5.11; N, 13.50.

Example 112-[5(6)-N-isopropylamidino-2-benzimidazoyl]-5-(4-aminophenyl)furan

The mono hydrochloride salt of the above nitro analog (0.411 g, 0.001mol) and 0.3 g of 10% Pd/C in 120 ml of methanol was subjected tohydrogenation at 50 psi for 4 h. The catalyst was removed by filtrationover filteraid, washed with warm methanol. The solvent volume wasreduced to approximately half under reduced pressure. The flaskcontaining the solution was placed in an ice bath and saturated with HClgas. The mixture was stirred at room temperature for 4 h and treatedwith dry ether and the solid was collected by filtration. The solid wasdried under vacuum at 80° C. for 24 h to yield 0.41 g (87%) mp>300° C.¹H NMR (DMSO-d₆/D₂0): 8.04 (d, J=1.6 Hz, 1H), 7.91 (d, J=8.4 Hz 2H),7.80 (d, J=8.4 Hz 1H), 7.64 (dd, J=1.6,8.4 Hz, 1H), 7.60 (d, J=4.0 Hz,1H), 7.24(d, J=8.4 Hz, 2H), 7.14 (d, J=4.0 Hz 1H), 4.05 (septet, J=6.4Hz, 1H), 1.3(d, J=6.4 Hz, 6H). ¹³C NMR (DMSO-d₆): 162.4, 156.8, 144.4,140.9, 138.8, 137.6, 135.0, 126.3, 125.4, 124.6, 124.1, 121.1, 118.0,115.6, 114.9, 108.6, 45.6, 21.3. FABMS m/e 360(M⁺+1). Anal. Calcd forC₂₁H₂₁N₅O₃.3HCl: C, 53.80; H, 5.15; N, 14.93. Found: C, 54.22; H, 4.75;N, 15.05.

Example 12 2,5-Bis (2-Benzimidazolyl-4-cyanophenyl)furan

A mixture of 5-[4-cyanophenyl]-2-furancarboxaldehyde (1.97, 0.01 mol),1,2-phenylenediamine (1.06 g, 0.01 mol) and 1,4-benzoquinone (1.08 g,0.01 mol) in 50 ml dry ethanol was heated at reflux (under nitrogen) for8 h. The reaction mixture was cooled and diluted with ether andfiltered. The solid was collected and stirred with 1:3 mixture of EtOHand ether for 20 min and the yellow brown solid was filtered, washedwith ether and dried in vacuum at 70° C. for 12 h. which yielded 1.96 g(69%), mp 227–8° C. dec, ¹H-NMR(DMSO-d6): 8.06 (d, 2H, J=8.8 Hz),7.91(d, 2H, J=8.8 Hz), 7.60 (dd, 2H, J=3.2 Hz, J=6.4 Hz), 7.38 (d, 1H,J=3.6 Hz), 7.32(d, 1H, J=3.6 Hz), 7.23 (dd, 2H, J=3.2 Hz, J=6.4 Hz).NMR(DMSO-d6): 152.1, 146.0, 142.7, 138.7, 133.2, 132.6, 124.1, 122.3,118.4, 114.9, 112.5, 111.1, 109.8, MS: m/e 285 (M+). Anal. calcd. for:C18H11N3O: C, 75.79; H, 3.86; N, 14.73. Found: C, 75.88; H, 3.77; N,14.55.

2,5-Bis[2-Benzimidazolyl-4-(amidino) phenyl]furan dihydrochloride

The above cyano compound (2.85 g, 0.01 mol) in 60 ml ethanol wassaturated with dry HCl gas at 0–5° C. The reaction mixture was stirredat room temperature for 12 days (monitored by IR and TLC). The mixturewas diluted with ether and the yellow imidate ester hydrochloride wasfiltered, washed with ether and dried under vacuum for 6 h 3.73 g (92%).The solid was used in next step without further purification. Asuspension of the imidate ester hydrochloride (0.808 g, 0.002 mol) in 35ml ethanol was saturated with ammonia gas at 0–5° C. and stirred for 24h at room temperature. The solvent was reduced to one-third underreduced pressure, diluted with ether and filtered. The yellow solid wasresuspended in 10 ml ethanol and treated with 4 ml saturated ethanolicHCl and stirred at 35° C. for 2 h. The solvent was removed under vacuumand the residue triturated with ether, filtered, washed with ether anddried under vacuum at 45° C. for 24 h to yield 0.61 g (81%) yellow solidmp>280° C. dec. ¹H-NMR(DMSO-d6/D2): 8.15(d, 2H, J=8.7 Hz), 7.93 (d, 2H,J=8.7 Hz), 7.78 (d, 1H, J=3.6 Hz), 7.75 (dd, 2H, J=3 Hz, J=6.3 Hz), 7.50(d, 1H, J=3.6 Hz), 7.49 (dd, 1H, J=3 Hz, J=6.3 Hz). ¹³C-NMR(DMSO-d6):165.0, 155.9, 139.8, 139.7, 133.4, 132.4, 129.3, 127.8, 126.3, 125.2,119.5, 114.3, 112.0. FABMS: m/e 303 (M++1). Anal. calcd. for:C18H14N4O.2HCl: C,57.61; H,4.29; N, 14.93. Found; C, 57.45; H, 4.46; N,14.64.

2,5-Bis[2-Benzimidazolyl-4-(2-imidazolino) phenyl]furan dihydrochloride

A mixture of the imidate ester hydrochloride (0.808 g, 0.002 mol) fromabove, ethylenediamine (0.12 g, 0.002 mol) in 20 ml of dry ethanol washeated at reflux for 12 h. The solvent volume was reduced to 8 ml underreduced pressure and diluted with ether. The resultant solid wasfiltered and dried. This solid was dissolved in 35 mL hot ethanol andsaturated with HCl gas at room temperature. The mixture was stirred at50° C. for 2 h and concentrated under reduced pressure and 30 ml dryether was added. The precipitated yellow salt was filtered, washed withether and dried under vacuum at 70° C. for 24 h to yield 0.69 g (84%)yellow solid mp>300° C. dec. ¹H-NMR(DMSO-d6/D2): 8.06(d, 2H, J=8.7 Hz),7.91 (d, 2H, J=8.7 Hz), 7.71 (dd, 2H, J=3 Hz, J=6 Hz), 7.64 (d, 1H,J=3.9 Hz), 7.47(dd, 1H, J=3 Hz, J=6.3 Hz), 7.44 (d, 1H, J=3.9 Hz), 3.94(s, 4H). ¹³C-NMR(DMSO-d6): 164.6, 155.7, 140.3, 140.1, 133.9, 132.9,129.7, 126.7, 125.4, 122.1, 119.2, 114.6, 112.5, 44.8, FABMS: m/e 303(M++1). Anal. calcd for: C20H16N4O.2HCl.0.5H2O: C,58.54; H,4.67; N,13.65. Found; C, 58.54; H, 4.67; N, 13.66.

EXAMPLES 13–24

Anti-BVDV Properties of Inventive Compounds

Example 13

Screening of antiviral compounds for anti-BVDV activity

2.0 cm² wells in a 24-well plate were seeded with 50 μl of medium from12 ml of MEM-eq (minimum essential medium (MEM) with Earle's saltssupplemented with 10% (v/v) equine serum, sodium bicarbonate (0.75mg/ml), L-glutamine (0.29 mg/ml), penicillin G (100 U/ml), streptomycin(100 μg/ml), and amphotericin B (0.25 μg/ml)), which was derived bytrypsinization of a confluent monolayer of Madin Darby Bovine Kidney(MDBK) cells in a 25 cm² flask. Cells were incubated at 38.5° C. with 5%CO₂ for 24 hours. The average number of cells per well was determinedand later used to calculate appropriate multiplicities of infection(MOI) of BVDV virus.

Cells were inoculated with BVDV in medium containing test antiviralcompounds (12.5 μM, 200 μL total volume), as follows:

two wells had no BVDV, and no antiviral compound

one well had BVDV at 0.05 MOI, and no antiviral compound

one well had BVDV at 1.0 MOI, and no antiviral compound

ten wells had BVDV at 0.05 MOI, and 12.5 μM of antiviral compound

ten wells had BVDV at 1.0 MOI, and 12.5 μM of antiviral compound.

The inoculated cells incubated for one hour at 38.5° C. with 5% CO₂ inhumidified air. The medium was removed from the wells, and the cellswashed one time with Ca²⁺ and Mg²⁺-free PBS comprising antiviralcompound (12.5 μM) (cells in the wells not initially treated withantiviral compounds were washed without antiviral compound). One ml ofMEM-eq comprising antiviral compound (12.5 μM) was added to wellsinitially treated with antiviral compound; those not treated withantiviral compound initially did not receive antiviral compound at thisstep. Three days post-inoculation, medium was removed and stored at −20°C. for assay. One ml of fresh medium containing 200 μL total (12.5 μM)antiviral compound was is added to wells initially treated withantiviral compound; those not treated with antiviral compound initiallydid not receive antiviral compound at this step. Seven dayspost-inoculation, medium was removed and stored at −80° C. for serialdilution & assay.

The MDBK cells were resuspended in MEM-eq with no antiviral compound.Uterine tubal cells (UTC) were freeze-thawed and stored at −80° C. foranalysis. UTC lysates were serially diluted with medium from Day 7 andassayed by immunoperoxidase for the presence of BVDV.

Example 14

Immunoperoxidase monolayer assay for BVDV

All samples were assayed for BVDV using the immunoperoxidase monolayerassay as described in A. Afshar et al., Can J Vet Res; 55:91–93 (1991).Samples were assayed in triplicate by adding 50 μL of MEM-eq containingapproximately 2.5×10³ MDBK cells to 50-μL of each sample supplementedwith 50 μL of fresh MEM-eq in a 96-well culture plate. Plates wereincubated for 72 h at 38.5° C. in a humidified atmosphere of 5% CO2 andair before the immunoperoxidase labeling technique was performed asfollows:

After fixation, potentially infected cells were incubated withmonoclonal antibodies D89 (M. L. Vickers et al., J Vet Diagn Invest 2,300–302 (1990); Xue W et al., J Clin Microbiol 28,1688–1693 (1990))specific for E2/gp53, a major envelope glycoprotein of BVDV (Xue W etal., Vet Microbiol 57,105–118 (1997)) and 20.10.6 specific for NS3-p80,a conserved nonstructural protein (W. V. Corapi et al., Am J Vet Res 51,1388–1394 (1990)). After washing with PBS and Tween 20 to remove unboundantibodies, peroxidase-conjugated rabbit anti-mouse IgG (Jackson ImmunoResearch Lab, West Grove, Pa.) was added. After a short incubationperiod, unbound peroxidase-conjugated antibody was removed by washingwith PBS and Tween 20. Finally, the enzyme substrate, aminoethylcarbazole (Zymed Laboratories, Inc., South San Francisco, Calif.), whichproduces a reddish-brown color when oxidized by horseradish peroxidase,was added. Color change was visualized under light microscopy andcompared to known positive and negative controls on each plate.

Example 15

Tissue Culture Passage

All samples other than stock virus aliquots were also passaged in tissueculture to optimize isolation of BVDV. Upon initial thawing, 200 μL ofeach sample was inoculated onto a 2 cm² well seeded 24 h previously withMDBK cells. Passages were incubated 5 days (d) at 38.5° C. in anatmosphere of 5% CO₂ and humidified air. Passages were frozen at −80° C.for storage. Tissue culture passage samples were thawed and assayed byvirus isolation if isolation of BVDV was unsuccessful from the originalsample. Samples were reported to be free of BVDV by virus isolation onlyif virus was not detected after each of two serial passages.

Example 16

Reverse Transcription Nested Polymerase Chain Reaction Assay (RT-nPCR)

A reverse transcription nested polymerase chain reaction assay fordetecting BVDV was performed on all samples other than stock virusaliquots. Upon initial thawing, RNA was isolated from samples using theQIAamp® viral RNA mini kit (Qiagen, Valencia, Calif.) according to themanufacturer's instructions. RNA samples were stored at −80° C. untilRT-nPCR was performed.

All steps of complementary DNA production (cDNA) and amplification werecarried out in a single closed-tube reaction using a modification of theprotocol of McGoldrick et al. (see Duffell S J et al., Vet Rec1985;117:240–245; Givens M D, et al., Theriogenology 2000;54:1093–1107;Lang-Ree JR, et al., Vet Rec 1994;135:412–413). In the first step, 5 μLof trehalose (22% w/v stock; Sigma, St Louis, Mo., cat #T5251) was usedto store and maintain the following mixture in the lid of a 200-μL,thin-walled tube: 0.4 μL of each inner primer BVD 180 and HCV 368 (50μM), 1 μL of dNTPs (10 mM) and 0.25 μL of Taq Polymerase (1.25 U,Promega, Madison, Wis.). The tubes were left to dry for 2 h at roomtemperature prior to storage.

In the second step, the initial reverse transcription polymerase chainreaction was performed in the bottom of the tubes containing the driedtrehalose mixture within the lid. Two μL of RNA were added through theoverlaid mineral oil (50 μL) to the initial reaction volume (48 μL)containing the following reagents (Promega): 5 μL 10× buffer, 8 μL ofMgCl2 (25 mM), 2 μL of dNTPs (10 mM), 1 μL of each outer primer BVD 100and HCV 368 (5 μM), 1 μL of Triton X-100 (100% stock), 0.25 μL ofdithiothreitol (100 mM), 0.25 μL (10 U) RNAsin, 0.5 μL (2.5 U) of Taqpolymerase, and 0.5 μL (100 U) of MMLV (Moloney Murine Leukemia Virus)reverse transcriptase. The tubes were then subjected to the followingcycle parameters: 37° C. for 45 min, 95° C. for 5 min and then 20 cyclesat 94° C. for 1 min, 55° C. for 1 min and 72° C. for 1 min.

A final elongation step of 72° C. for 10 min completed the initialamplification reaction. In the third step, the tubes were invertedseveral times to mix the samples in the lid and in the base to initiatethe nested polymerase chain reaction (nPCR). The tubes were thencentrifuged at 14,000×g for 12 sec before returning to the thermocyclerfor nPCR, using 30 cycles of 94° C. for 1 min, 55° C. for 1 min and 72°C. for 45 sec. A final elongation step of 72° C. for 10 min completedthe amplification process prior to maintaining the reactions at 4° C.Five microliter aliquots of PCR products were separated by 1.5% agarosegel electrophoresis. The agarose gels contained 0.5 μg/ml ethidiumbromide to allow visualization of RT-nPCR products using an ultraviolettransilluminator.

The outer primers, BVD 100 (5′-GGCTAGCCATGCCCTTAG-3′) (SEQ ID NO. 1) andHCV 368 (5′-CCATGTGCCATGTACAG-3′) (SEQ ID NO. 2) amplified a 290 basepair sequence of the 5′ untranslated region of the viral genome. Theinner primers, BVD 180 (5′-CCTGAGTACAGGGDAGT CGTCA-3′) (SEQ ID NO. 3)and HCV 368 amplified a 213 base pair sequence within the firstamplicon. The novel BVD 180 primer was degenerate at the 14th base(D=G+A+T) to accommodate differences within the 5′ untranslatedsequences of virus strains used in this research as determined byautomated dye terminator nucleotide sequencing (Nucleic Acid ResourceFacility, Auburn University, Ala.) of the initial PCR products fromviral stocks.

Example 17

Oocyte Collection and Maturation

Cow ovaries were collected at an abattoir in Omaha, Nebr., and placed inPBS for transport to a nearby laboratory. The contents of 1- to 10-mmfollicles were aspirated at a vacuum rate of 21.5 ml/min and poured ontoa 70 μm filter. Cellular components of the pooled follicular aspiratewere rinsed with TL-HEPES and searched for oocytes surrounded bymultiple layers of dense cumulus cells. Useable cumulus oocyte complexes(COCs) were washed two additional times in TL-HEPES, then placed in 7.5ml of maturation media that had previously equilibrated at 38.5° C. inan atmosphere of 5% CO₂ and humidified air. The maturation media wasthen sealed and maintained at 38.5° C. for 20 to 22 h while beingtransported to the experimental laboratory.

Example 18

Media for in vitro fertilization/embryo assays

Oocytes were matured in cell culture medium 199 (CCM 199) with Earle'ssalts (GIBCO-BRL, Grand Island, N.Y., USA) supplemented with 10% (v/v)heat-inactivated fetal bovine serum (FBS; HyClone Lab., Inc., Logan,Utah, USA), sodium pyruvate (11 μg/ml), bovine FSH (0.01 U/ml), bovineLH (0.01 U/ml), penicillin (100 U/ml) and streptomycin (100 μg/ml).

Matured oocytes were fertilized in CR2 medium (C. F. Rosenkrans et al.,Theriogenology 35, 266 (1991)) supplemented with BSA (6 mg/ml), heparin(10 μg/ml), penicillamine (0.3 μg/ml), hypotaurine (0.2 μg/ml),penicillin (100 U/ml) and streptomycin (100 μg/ml).

The first three days (d) of in vitro culture (IVC) was in CR2 mediumsupplemented with BSA (6 mg/ml), penicillin (100 U/ml) and streptomycin(100 μg/ml). The last four d of IVC was in CR2 medium supplemented with10% (v/v) FBS, penicillin (100 U/ml) and streptomycin (100 μg/ml).

Example 19

Exposure to Bovine Viral Diarrhea Virus (BVDV)

After in vitro maturation (IVM), COCs were washed 5 times in 3 ml ofMEM-eq. After washing, COCs were exposed to a noncytopathic strain ofBVDV in 3 ml of MEM-eq or maintained separately as negative controls inBVDV-free MEM-eq. Exposed and unexposed COCs were incubated for 1 h at38.5° C. in an atmosphere of 5% CO2 and humidified air, and then washed3 times in 3 ml of TL-HEPES before addition to IVF drops.

The noncytopathic strains of BVDV used in this research included 2diverse Genotype I strains (SD-1 and NY-1) and 2 diverse Genotype IIstrains (CD-87 and PA-131). Givens M D et al., Theriogenology2000;54:1093–1107. All stocks were propagated in BVDV-free MDBK cellscultured in MEM-eq. Virus was harvested by freezing and thawing and wasstored in cryovials at −80° C. until needed.

Example 20

In vitro Fertilization

Matured COCs were placed in 42-μL drops of fertilization medium undermineral oil. Cryopreserved bovine semen from a single collection wasused for fertilization. This semen was confirmed to be free of BVDV byvirus isolation and RT-nPCR. After PERCOLL®-gradient (45 to 90%)separation, 1.5×10⁵ spermatozoa were added to each fertilization drop,which was incubated for approximately 18 h at 38.5° C. in a humidifiedatmosphere of 5% CO₂ and air.

Example 21

In vitro Culture

After the IVF period, presumptive zygotes were removed, washed 4 timesin TL-Hepes, equilibrated in IVC medium with BSA, and placed withcumulus cells still attached in 30-μl drops (10 to 12 per drop) of theIVC medium with BSA under mineral oil. The IVC plates were incubated for3 d at 38.5° C. in a humidified atmosphere of 5% CO₂ and air. After thefirst 3 d in IVC, embryos were washed 3 times in TL-Hepes, and most ofthe cumulus cells were removed by gentle aspiration in and out of asterile pipette. The nearly nude embryos were examined for cleavage, andthose at the 5-cell stage or greater were washed 1 more time in IVCmedium and placed with pieces of detached cumulus in 60-μl drops (20 to25 per drop) of the IVC medium with 10% (v/v) FBS under mineral oil.These developed embryos were incubated an additional 4 d. After thefinal 4 d in IVC, embryos were transferred into 3 ml of MEM-eq,separated from cumulus cells, and development to the morula orblastocyst stage was noted.

Example 22

Washing and Trypsin Treatment of Embryos

Washing and trypsin treatment of Day 7 embryos conformed to proceduresrecommended by the International Embryo Transfer Society for treatmentof in vivo-derived bovine embryos. (Stringfellow D A, et al., Manual ofthe International Embryo Transfer Society Third Edition., Savoy Ill.:International Embryo Transfer Society, 1998;79–84). Degenerate anddeveloped Day 7 embryos were washed 12 times in 1 ml of MEM-eq in 2-cm2wells.

For trypsin treatment, twelve 3-ml washes in 35-mm Petri dishes wereused. The first 5 and last 5 washes were PBS supplemented with 0.4% BSA,penicillin (100 U/ml) and streptomycin (100 μg/ml). The 6th and 7thwashes were trypsin diluted 1:250 in 3 ml of Hank's balanced saltsolution without Ca²⁺ and Mg²⁺. Embryos were treated in trypsin forapproximately 90 sec (45 sec/wash) before proceeding through the last 5washes.

Example 23

Samples assayed for BVDV

During each of 12 research replicates (3 replicates with 4 diversestrains of BVDV), 140 to 180 COCs were exposed to virus while 50 to 80COCs were maintained as negative controls. For each replicate, sampleswere obtained from exposed and unexposed cultures to be assayed forBVDV. All samples other than stock virus aliquots were assayed for BVDVusing virus isolation with (a) immunoperoxidase assay for viraldetection, (b) tissue culture passage prior to virus isolation tooptimize viral detection, and (c) RT-nPCR. Samples included:

Stock virus aliquots. The viral aliquot to which the COCs were exposedwas serially diluted and assayed by virus isolation usingimmunoperoxidase assay.

Day 3 cumulus cells. On Day 3 of IVC, some detached cumulus cells wereremoved from the 3rd wash of TL-Hepes, transferred into 3 ml of MEM-eq,and then placed in 500 μL of MEM-eq within a cryovial. Cells were lysedby freezing at −80° C. and thawing to release any intracellular virusprior to assay for BVDV.

Day 7 cumulus cells. On Day 7 of IVC, cumulus cells from exposed andunexposed cultures were transferred from the 3 ml of MEM-eq into 500 μLof MEM-eq within a cryovial. Cells were lysed by freezing at −80° C. andthawing to release any intracellular virus prior to assay for BVDV.

Day 7 individual embryos. If sufficient numbers of BVDV-exposed M/Bdeveloped by Day 7 of each research replicate, a group of 10 M/B waswashed as previously described and a group of 10 M/B was trypsin-treatedas previously described. Virus-exposed, washed M/B were individuallyplaced into 500 μL of MEM-eq (4 to 5 per replicate) or were individuallycryopreserved and thawed before placement in MEM-eq (4 to 5 perreplicate). Virus-exposed, trypsin-treated M/B were individually placedinto 500 μL of MEM-eq (3 to 5 per replicate) or were individuallycryopreserved and thawed before placement in MEM-eq (4 to 5 perreplicate). All samples were sonicated before viral assay.

If sufficient numbers of non-exposed M/B developed by Day 7 of eachreplicate, a group of 10 M/B was washed as previously described.Non-exposed, washed M/B were individually placed directly into 500 μL ofMEM-eq (5 per replicate) or were individually cryopreserved and thawedbefore placement in MEM-eq (5 per replicate). Samples were sonicatedbefore viral assay.

Example 24

Statistical Analysis and Results

The tissue culture infective dose 50% (TCID₅₀)/ml of the exposurealiquot was determined by the method of Reed and Muench (L. J. Reed andH. Muench, Am J Hygiene 27, 493–497 (1938). Results of viral detectionassays were compared using a Pearson Chi-square test statistic (J. Salland A. Lehman, JMP Start Statistics (Duxbury Press, Belmont, Calif.(1996),195–211).

Table 2 sets forth the results of the analysis of in vitro culture mediaand cell lysates that have been treated with the indicated antiviralcompound for the indicated time at a concentration of 12.5 μM, afterexposure to BVDV at a MOI of 0.05 (see Example 12).

TABLE 2 Day 3 Media Day 7 Media Day 7 Cell Lysates Antiviral drugTCID₅₀/mL % Control TCID₅₀/mL % Control TCID₅₀/mL % Control No antiviral2.00E+05 5.20E+05 2.00E+06 DB619 3.50E+02 0.18% 2.00E+05 38.46% 1.10E+0655.00% DB673 1.00E+02 0.05% 6.20E+03  1.19% 3.50E+04  1.75%

Table 3 sets forth the results of the analysis of in vitro culture mediaand cell lysates that have been treated with the indicated antiviralcompound for the indicated time at a concentration of 12.5 μM, afterexposure to BVDV at a MOI of 1.0 (see Example 12).

TABLE 3 Day 3 Media Day 7 Media Day 7 Cell Lysates Antiviral drugTCID₅₀/mL % Control TCID₅₀/mL % Control TCID₅₀/mL % Control No antiviral3.50E+05 3.50E+04 6.20E+05 DB619 6.20E+04 17.71% 3.50E+04 100.00%3.50E+05 56.45% DB673 3.50E+02  0.10% 2.00E+05 571.43% 2.70E+05 43.55%

Table 4, below sets forth the results of the analysis of Day 3 in vitroculture media and Day 3 cell lysate that has been treated with theindicated antiviral compound at a concentration of 12.5 μM, afterexposure to BVDV at a MOI of 0.5 (see Example 12).

TABLE 4 Day 3 Media Day 3 Cell Lysates Antiviral drug TCID₅₀/mL %Control TCID₅₀/mL % Control No antiviral 3.50E+06 6.20E+06 DB 4571.00E+02 0.0029% 3.50E+02 0.0056% DB 458 Negative Negative DB 459Negative 1.00E+02 0.0016% DB 606 Negative Negative DB 680 NegativeNegative DB 701 6.20E+03 0.1771% 3.50E+04 0.5645% DB 705 NegativeNegative DB 708 Negative Negative DB 711 2.40E+05 6.8571% 2.00E+07322.5806% DB 752 Negative 6.20E+02 0.01%

Table 5 sets forth the results of the analysis of Day 3 cell lysatesthat have been treated with the indicated antiviral compound for threedays at the indicated concentration, after exposure to BVDV at a MOI of0.05.

TABLE 5 Day 3 Media Day 3 Cell Lysates Antiviral drug TCID₅₀/mL %Control TCID₅₀/mL % Control No antiviral 2.00E+06 3.50E+05 DB 456 25 μm3.50E+03 0.1750% 2.00E+03 0.5714% DB 456 12 μm 6.20E+02 0.0310% 3.50E+031.0000% DB 456 6 μm 3.50E+04 1.7500% 3.50E+04 10.0000% DB 456 3 μm3.50E+05 17.5000% 6.20E+05 177.1429% DB 456 1.5 μm 5.10E+05 25.5000%3.50E+06 1000.0000% DB 456 0.7 μm 6.30E+05 31.5000% 3.50E+06 1000.0000%DB 456 0.4 μm 6.30E+05 31.5000% 3.50E+06 1000.0000% DB 456 0.2 μm6.30E+05 31.5000% 3.50E+05 100.0000%

In the specification, and examples there have been disclosed typicalpreferred embodiments of the invention and, although specific terms areemployed, they are used in a generic and descriptive sense only and notfor the purposes of limitation, the scope of the invention being setforth in the following claims.

1. A compound according to Formula I:

wherein: X₁ is O; X₂ is CH; X₃ is NR₉, wherein R₉ is H or alkyl; X₄ isN; A is selected from the group consisting of

R₁, R₂, R₃, and R₄ are each independently selected from the groupconsisting of H, alkyl, alkoxy, halide, alkylhalide, nitro and NR₁₀R₁₁,wherein R₁₀ and R₁₁ are independently selected from H and lower alkyl;R₆ is H, alkyl or aryl; and R₇ and R₈ are each independently selectedfrom the group consisting of H and alkyl.
 2. The compound according toclaim 1, wherein: X₃ is NH and R₂, R₃ and R₄ are each H.
 3. The compoundaccording to claim 1, wherein A is

and R₆ is alkyl.
 4. The compound according to claim 1, wherein A is

and R₇ and R₈ are each H.
 5. The compound according to claim 1, whereinR₁ is —NR₁₀R₁₁, wherein R₁₀ and R₁₁ are independently selected from Hand lower alkyl.
 6. The compound according to claim 1, wherein R₁ is anitro Croup.
 7. The compound according to claim 1, wherein the compoundis represented by the formula:


8. The compound according to claim 1, wherein the compound isrepresented by the formula:


9. The compound according to claim 1, wherein the compound isrepresented by the formula:


10. The compound according to claim 1, wherein the compound isrepresented by the formula:


11. A pharmaceutical composition comprising a compound of claim 1, in apharmaceutically acceptable carrier.
 12. The pharmaceutical compositionaccording to claim 11, wherein the composition is formulated forintravenous administration.
 13. The pharmaceutical composition accordingto claim 11, wherein the composition is formulated for oraladministration.
 14. A compound according to Formula II:

wherein: X₁ is O; X₂ is CH; X₃ is NR₉, wherein R₉ is H or alkyl; X₄ isN; A is selected from the group consisting of

R₁, R₂, and R₃ are each independently selected from the group consistingof H, alkyl, alkoxy, halide, alkylhalide, nitro and NR₁₀R₁₁, wherein R₁₀and R₁₁ are independently selected from H and lower alkyl; R₆ is H,alkyl or aryl; and R₇ and R₈ are each independently selected from thegroup consisting of H and alkyl.
 15. A pharmaceutically compositioncomprising a compound of claim 14, in a pharmaceutically acceptablecarrier.
 16. The pharmaceutical composition according to claim 15,wherein the composition is formulated for intravenous administration.17. The pharmaceutical composition according to claim 15, wherein thecomposition is formulated for oral administration.
 18. A compoundaccording to Formula III:

wherein: X₁ is O; X₂ is CH; X is NR₉, wherein R₉ is H or alkyl; X₄ is N;A is selected from the group consisting of

R₁, R₂, R₃, and R₄ are each independently selected from the groupconsisting of H, alkyl, alkoxy, halo, amidine, nitro and NR₁₀R₁₁ whereinR₁₀ and R₁₁ are independently selected from H and lower alkyl; R₆ is H,alkyl or aryl; and R₇ and R₈ are each independently selected from thegroup consisting of H and alkyl.
 19. A pharmaceutical compositioncomprising a compound of claim 18, in a pharmaceutically acceptablecarrier.
 20. The pharmaceutical composition according to claim 19,wherein the composition is formulated for intravenous administration.21. The pharmaceutical composition according to claim 19, wherein thecomposition is formulated for oral administration.
 22. A compoundaccording to Formula IV:

wherein: X₁ is O; X₂ is CH; X₃ is NR₉, wherein R₉ is H or alkyl; X₄ isN; A is selected from the group consisting of

R₁, R₂, and R₃ are each independently selected from the group consistingof H, alkyl, alkoxy, halide, alkylhalide, amidine, nitro and NR₁₀R₁₁,wherein R₁₀ and R₁₁ are independently selected from H and lower alkyl;R₆ is H, alkyl or aryl; and R₇ and R₈ are each independently selectedfrom the group consisting of H and alkyl.
 23. A pharmaceuticalcomposition comprising a compound of claim 22, in a pharmaceuticallyacceptable carrier.
 24. The pharmaceutical composition according toclaim 23, wherein the composition is formulated for intravenousadministration.
 25. The pharmaceutical composition according to claim23, wherein the composition is formulated for oral administration.
 26. Amethod of treating bovine viral diarrhea virus (BVDV) infection in asubject in need of such treatment, comprising administering to thesubject a compound selected from the group consisting of Formula I andFormula II:

wherein: X₁ is O: X₂ is CH; X₃ is NR₉, wherein R₉ is H or alkyl: X₄ isN; A is selected from the group consisting of

R₁, R₂, R₃, and R₄ are each independently selected from the groupconsisting of H, alkyl, alkoxy, halide, alkylhalide, nitro and —NR₁₀R₁₁,wherein R₁₀ and R₁₁ are independently selected from H and lower R₆ is H,alkyl or aryl; and R₇ and R₈ are each independently selected from thegroup consisting of H and alkyl; or a pharmaceutically acceptable saltthereof, in an amount sufficient to treat the bovine viral diarrheavirus (BVDV) infection.
 27. The method according to claim 26, whereinthe compound is a compound of Formula I.
 28. The method according toclaim 26, wherein the compound is represented by the formula:


29. The method according to claim 26, wherein the subject is a cow. 30.The method according to claim 26, wherein the subject is an embryo. 31.The method according to claim 26, wherein the compound is administeredintravenously.
 32. The method according to claim 26, wherein thecompound is administered orally.
 33. A method of treating bovine viraldiarrhea virus (BVDV) infection in a subject in need of such treatment,comprising administering to the subject a compound selected from thegroup consisting of Formula III and Formula IV:

wherein: X₁ is O; X₂ is CH: X₃ is NR₉, wherein R₉ is H or alkyl; X₄ isN; A is selected from the group consisting of

R₁, R₂, R₃, and R₄ are each independently selected from the groupconsisting of H, alkyl, alkoxy, halide, alkylhalide, amidine, nitro and—NR₁₀R₁₁, wherein R₁₀ d R₁₁, are independently selected from H and loweralkyl; R₆ is H, alkyl or aryl; and R₇ and R₈ are each independentlyselected from the group consisting of H and alkyl; or a pharmaceuticallyacceptable salt thereof, in an amount sufficient to treat the bovineviral diarrhea virus (BVDV) infection.
 34. A method of treatingFlaviviridae-related hepatitis C infection in a subject in need of suchtreatment, comprising administering to the subject a compound selectedfrom the group consisting of Formula I and Formula II:

wherein: X₁ is O; X₂ is CH; X₃ is NR₉, wherein R₉ is H or alkyl; X₄ isN; A is selected from the group consisting of

R₁, R₂, R₃ and R₄ are each independently selected from the groupconsisting of H, alkyl, alkoxy, halide, alkylhalide, nitro and —NR₁₀R₁₁,wherein R₁₀ and R₁₁ are independently selected from H and lower alkyl;R₆ is H, alkyl or aryl; and R₇ and R₆ are each independently selectedfrom the group consisting of H and alkyl; or a pharmaceuticallyacceptable salt thereof, in an amount sufficient to treat the hepatitisC infection.
 35. The method according to claim 34, wherein the compoundis a compound of Formula I.
 36. The method according to claim 34,wherein the compound is represented by the formula:


37. The method according to claim 34, wherein the subject is a human.38. The method according to claim 34, wherein the compound isadministered intravenously.
 39. The method according to claim 34,wherein the compound is administered orally.
 40. A method of treatingFlaviviridae-related hepatitis C infection in a subject in need of suchtreatment, comprising administering to the subject a compound selectedfrom the group consisting of Formula III and Formula IV:

wherein: X₁ is O; X₂ is CH; X₃ is NR₉, wherein R₉ is H or alkyl; X₄ isN; A is selected from the group consisting of

R₁, R₂, R₃, and R₄ are each independently selected from the groupconsisting of H, alkyl, alkoxy, halide, alkylhalide, amidine, nitro and—NR₁₀R₁₁, wherein R₁₀ and R₁₁ are independently selected from H andlower alkyl; R₆ is H, alkyl or aryl; and R₇ and R₈ are eachindependently selected from the group consisting of H and alkyl; or apharmaceutically acceptable salt thereof, in an amount sufficient totreat the hepatitis C infection.